Building cytoskeletal circuits with branched microtubule networks

Introduction: Cytoskeletal elements self-organize into intricate hierarchical structures, forming the molecular basis for various cellular functions. A remarkable example is the axonal microtubule (MT) architecture, crucial for controlled neuronal migration, axon extension, and long-distance molecular transport. To harness this natural phenomenon, we have developed cytoskeletal circuits as controllable platforms to engineer robust MT architectures. These circuits integrate the branching MT nucleation pathway with microfabrication techniques, enabling the adaptive self-organization of uniformly polarized MT arrays within microfluidic confinements. Our microfluidic structures provide control over this self-organizing process, allowing us to construct and characterize various elements like turns, divisions, biased divisions, and MT diodes, resulting in diverse MT architectures on a chip. Our work holds potential for the development of novel on-chip nanotechnologies.

Materials and

Methods: Protein Purification and Xenopus Egg Extract: EB1-GFP and RanQ69L proteins were purified using specific procedures involving bacterial expression, cellular lysis, affinity chromatography, and gel filtration. Xenopus egg extract, supplemented with labeled tubulin, EB1, RanQ69L, and inhibitors, was prepared for MT nucleation reactions.

Microfabrication Process: Microfluidic chips were fabricated by bonding PDMS blocks with microstructure-featured coverslip glass.
Coverslip glasses were cleaned, primed, and patterned with photoresist, while PDMS blocks containing microchannels were created through soft-lithography techniques.The bonding of PDMS and coverslip glass was achieved through air plasma treatment, resulting in microfluidic chips with multiple microchannels.

Microscopy and Image Processing: TIRF microscopy equipped with a high-resolution camera was used for image capture.
Kymographs and MT curvature were analyzed by manual extraction of shape and calculation of curvature and arc length.
EB1 comets on MT plus ends were detected by background removal, Gaussian filtering, and trajectory analysis using TrackMate, followed by custom MATLAB codes for further analysis. MT flux in different regions of interest was measured by counting EB1 comets over time.
This comprehensive approach allowed for the study of branching MT nucleation reactions within a controlled microfluidic environment

Results, Conclusions, and Discussions: We have developed cytoskeletal circuits as a platform for engineering controlled microtubule (MT) architectures. These circuits utilize branching MTs, whose organization is regulated by the microstructure of a microfluidic chamber. We have outlined the basic principles of this self-organization and have demonstrated its potential for designing complex MT architectures. We propose using biomolecular motors like kinesin-1 to enable robust on-chip molecular transport on these branching MTs, suggesting that the uniform polarity and high density of MTs within these circuits could enhance transport efficiency. Additionally, by integrating cytoskeletal circuits with other components, such as chemical, optical, magnetic, and electrical elements, we could create more complex structures like cytoskeletal switches and logic gates. We also discuss the potential applications beyond MT architecture, including the use of branching MTs for generating directed forces to manipulate micron/nano-sized objects and for nano-scale mechano-sensing within the circuits. Furthermore, we suggest that these circuits could be used to study how physical environments within cells and MT-associated proteins contribute to MT architecture in biological contexts, such as axons or fungal hyphae navigating confined spaces.

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